Reconstituting viral glycoproteins into large phospholipid vesicles

ABSTRACT

The present disclosure relates to novel liposome compositions and methods for their preparation. Utilization of the present invention provides an efficient reconstitution of membrane proteins into large (0.1 to 2 micron diameter) phospholipid vesicles with a large, internal aqueous space. The method has been exemplified with the use of glycoproteins of influenza (A/PR8/34) and Sendai (parainfluenza type I) viruses. The method comprises (A) extracting out the desired membrane protein from a source biological material with an extraction buffer comprising a detergent; (B) mixing the extract with a phospholipid solution and deriving a cochleate intermediate; and (C) forming large phospholipid vesicles with integrated membrane protein in a biologically active state.

This is a division of application Ser. No. 725,601 filed Apr. 22, 1985,(expected to issue on May 5, 1987) U.S. Pat. No. 4,663,161.

BACKGROUND OF THE INVENTION

Isolation and reconstitution into the bilayer of lipid vesicles orliposomes is one of the most powerful techniques applied to the study ofmembrane proteins. The simplicity of the resulting model system and theability to control experimental conditions, allow studies which would bedifficult or impossible to perform or interpret using intact cells,viruses, or whole isolated membranes. The structure and function ofviral glycoproteins and transport systems, and interactions of membranereceptors with lectins, viruses, and hormones have been investigatedusing liposome-reconstituted membrane proteins. This approach has alsoproven useful in a variety of immunological studies involving antibodyand complement binding, the activities and generation of cytotoxic Tlymphocytes, particle uptake by macrophages and the production ofsubunit vaccines.

Another reason for reconstituting membrane proteins into artificiallipid bilayers is to modify the properties of liposomes. Liposomes havetremendous potential as delivery vehicles in vivo and in vitro owing totheir ability to encapsulate, store, and transport materials. However,they have a low efficiency of attachment to cells. Also, a significantproportion of those which do attach are taken up by endocytosis, whichresults in their contents being delivered to lysosomes. This isdesirable in some cases, as in the proposed treatment of some lysosomalstorage diseases by liposomally delivered enzymes. However, if the goalis delivery to the cytoplasm or nucleus in a biologically active form,contact with the lysosomes should be avoided.

In enveloped viruses, the delivery of the viral nucleocapsid to thecytoplasm of the cell in a biologically active form is achieved throughthe interaction of the viral envelope glycoproteins with plasma membranecomponents. The viral glycoproteins mediate attachment to cell surfacereceptors and bring about the fusion of the viral envelope with the cellmembrane at the surface, or within the low pH environment of theendocytic vesicle. It has been suggested that liposomes containing thesebiologically active glycoproteins integrated in their lipid bilayermight be superior delivery vesicles. See in this regard Straubinger etal., Cell 32, 1069 (1983) and Volksy and Loyter, FEBS Lett. 92, 190(1978). It has been shown that reconstituted Sendai virus envelopes canefficiently deliver entrapped molecules to the cytoplasm and nucleus ofanimal cells. See in this regard Loyter and Volksy in MembraneReconstitution, Cell Surface Reviews (Poste, G. and Nicholson, G., eds.)Vol. 8, pp. 216-265 (1982).

The preparation of large unilamellar, phospholipid vesicles made by theuse of a calcium-EDTA-chelation technique has been described byPapahadjopoulos et al., Biochim. Biophys. Acta 394, 483 (1978).

However, liposomes prepared in accordance with the teaching of thisreference have not been heretofor used to reconstitute membrane proteinsin biologically active form. Moreover, the efficiency of encapsulationof materials within the liposome is relatively low.

SUMMARY OF THE INVENTION

The present invention relates to novel liposome compositions and methodsfor their preparation. Utilization of the present invention provides anefficient reconstitution of membrane proteins into large (0.1 to 2micron diameter) phospholipid vesicles with a large, internal aqueousspace. The method has been exemplified with the use of glycoproteins ofinfluenza (A/PR8/34) and Sendai (parainfluenza type I) viruses. Thetight association of the proteins with the lipid bilayer was indicatedby their migration with the phospholipid to the top of metrizamidedensity gradients after sonication, whereas encapsulated FITC-Dextranremained at the bottom of the gradient. Negative staining and electronmicroscopy showed peplomers on the surface of the vesicles which areindistinguishable from those seen on the respective viruses. Thereconstituted membrane proteins have been shown to retain theirbiological activities of receptor binding and membrane fusion as assayedby their ability to agglutinate and lyse red blood cells. In a preferredaspect, the method of the invention utilizes the nonionic detergent β-D-octyl-glucopyranoside, which is rapidly and easily removed as the meansof extracting the membrane proteins from the viral particles. Thisprocedure does not involve exposure to organic solvents, sonication, orextremes of pH, temperature, or pressure.

In a further preferred aspect of the present invention, the lipidvesicles are formed by use of rotary dialysis of the cochleateintermediates against a calcium chelating agent such as EDTA, EGTA,carbonate, citrate and the like.

DESCRIPTION OF THE INVENTION

In its broadest aspect, the present invention comprises a method for theefficient reconstitution of membrane proteins into large phospholipidvesicles with large, internal aqueous spaces. In the initial step ofthis method, a desired membrane protein is extracted out from the sourceparticle, cell, tissue, or organism utilizing an extraction buffercontaining a detergent which does not destroy the biological activity ofthe membrane protein. Suitable detergents include ionic detergents suchas cholate salts, deoxycholate salts and the like or nonionic detergentssuch as those containing polyoxyethylene or sugar head groups orheterogeneous polyoxyethylene detergents such as Tween or Brig orTriton. Preferred detergents are nonionic detergents containing sugarhead groups such as the alkyl glucosides. A particularly preferrednonionic detergent for this purpose is β-D-octyl-glucopyranoside.Utilization of this method allows efficient reconstitution of themembrane proteins into the liposomes with retention of biologicalactivities. This step avoids previously utilized organic solvents,sonication, or extremes of pH, temperature, or pressure, all of whichmay have an adverse effect upon efficient reconstitution in abiologically active form of the desired membrane proteins.

The buffer component utilized in conjunction with the aforesaiddetergents can be any conventional buffer employed for membrane proteinextractions. A suitable extraction buffer for the present purposes canbe prepared utilizing a 2MNaCl, 0.02M sodium phosphate buffer (pH 7.4).The concentration of the detergent component in the buffer is notnarrowly critical and can be in the range of from 0.1 to 20% (w/v)preferably from 1 to 5%, most preferably about 2%. The extractionefficiency can be enhanced by utilizing techniques well known in theart, such as by vortexing and sonicating. The extracted membraneproteins can be removed from the remaining nonsoluble debris byprocedures well known in the art, such as for example by centrifugation.The resulting supernatant containing the extracted membrane protein maythen be applied directly in the liposome formation procedure.

Membrane proteins which can be employed in the practice of the presentinvention include viral proteins such as for example viral envelopeprotein, animal cell membrane protein, plant cell membrane protein,bacterial membrane protein, parasite membrane protein, viral membraneprotein and the like. These respective proteins can be separated fromother components by procedures well known in the art prior tointroduction into the present methodology or they can be resolved duringthe course of the procedure as will be described below.

Suitable viruses which can be employed in conjunction with the method ofthe invention include Sendai, influenza, herpes simplex or genitalis,HTLV I, II or III, retroviruses, pox virus, respiratory syncytial virus,toga virus, and the like. The present invention can also be employed inconjunction with membrane proteins derived from bacterial or parasiticorganisms such as for example organisms causing malaria, chlamydia,N.gonorrhea, salmonella, liver flukes and the like.

The reconstituted viral, bacterial or parasitic membrane proteins in theimproved liposome compositions of the present invention can be employedas vaccines to render immunity to hosts treated with such compositions.

In the next step of the method of the present invention, the aforesaidextracted membrane proteins are mixed with phospholipid to form acochleate intermediate. There are several known procedures for makingsuch cochleates. One such method is the so-called standard cochleateobtained by use of the calcium-EDTA-chelation technique described byPapahadjopoulos et al., supra. In an embodiment of the presentinvention, a modification of such procedure is employed. In suchmodified procedure a negatively charged phospholipid such asphosphatidylserine, phosphatidic acid or phosphatidyl glycerol in theabsence or presence of cholesterol (up to 3:1, preferably 9:1 w/w) areutilized to produce a suspension of multilamellar protein lipid vesicleswhich were converted to small unilamellar protein lipid vesicles bysonication under nitrogen. These vesicles are dialyzed at roomtemperature against buffered calcium chloride resulting in the formationof an insoluble precipitate referred to as a cochleate cylinder. Aftercentrifugation, the resulting pellet can be taken up in buffer to yieldthe cochleate solution utilized in the preparation of the liposomes ofthe present invention.

In an alternative and preferred embodiment, an amount ofphosphotidylserine and cholesterol in the same proportions as above andequal to from about 1 to 10 times the weight, preferably equal to fourtimes the weight of the viral lipid were utilized to prepare thecochleates. Supernatant from the nonionic detergent extraction of themembrane proteins was then added and the solution vortexed for fiveminutes. This solution was then dialyzed against buffered calciumchloride to produce a precipitate which can be called a DC (for directcalcium dialysis) cochleate.

An additional, related method for reconstituting membrane proteins intoliposomes has been developed. The initial steps involving addition ofextracted viral envelope to dried down phosphatidylserine andcholesterol are the same as for the DC method. However, the solution isnext dialyzed against Buffer A (with no calcium) to form small liposomescontaining the glycoproteins. Calcium is then added either directly orby dialysis to form a precipitate. The precipitate is pelleted andvesicles are formed by direct addition of EDTA or rotary dialysis. Thesevesicles have physical characteristics and biological activities similarto those formed by the DC method, except that LC's are somewhat smallerand have lower encapsulation efficiencies.

The formation of vesicles from the above intermediates can be carried byalternative methodologies. In one procedure, the aforesaid cochleateswere pelleted by centrifugation at 60,000 xg at 5° C. The supernatantswere removed and replaced by a small quantity of buffer containing thematerial to be encapsulated. The pellets were then resuspended byvortexing. Five microliter aliquots of 150 mM EDTA (pH 9.5) are addedwith gentle mixing and frequent monitoring of pH. It is desired tomaintain pH to near neutrality during the vesicle formation procedure.When the pH became slightly basic, 150 mM EDTA (pH 7.5) was added untilthe cochleates were dissolved and an opalescent suspension of vesicleswas obtained.

In an alternative but preferred embodiment, rotary dialysis wasemployed. In this procedure, the supernatants were removed andtransferred to a small segment of dialysis tubing. The dialysis tubinghad been previously boiled with a sodium carbonate solution and thenwith distilled water, cut to the appropriate length and autoclaved indistilled water just prior to use. Small aliquots of buffer (5 to 10microliters) containing material to be encapsulated were used to riseout the tube and quantitatively tranfer the cochleates to the dialysisbag. The samples were dialyzed by rotating at room temperature againstbuffered 10 mM EDTA (final pH 7.4) until the cochleate precipitatedissolved and an opalescent suspension of vesicles was obtained.

The methodology employed in the practice of the present invention ismore specifically described in the examples which are set forth below.

EXAMPLES Materials and Methods

Materials. Bovine brain phosphatidylserine in chloroform was purchasedfrom Avanti Polar Lipids, Birmingham, Ala in glass ampules and storedunder nitrogen at -20° C. Cholesterol (porcine liver) grade I,n-octyl-β-D-glucopyranoside, fluorescein isothiocyanate (FITC)-dextran(average mol. wt. 67,000), metrizamide grade I, and chemicals forbuffers and protein and phosphate determinations, were obtained fromSigma Chemical Company, St. Louis, Mo. Organic solvents were purchasedfrom Fisher Scientific Co., Fairlawn, NJ. Reagents for polyacrylamidegel electrophoresis were from BioRad Laboratories, Richmond, Ca. S1000Sephacryl Superfine was obtained from Pharmacia, Piscataway, NJ. Thickwalled polycarbonate centrifuge tubes (10 ml capacity) from BeckmanInstruments, Palo Alto, Ca, were used for vesicle preparations, washes,and gradients. A bath type sonicator, Model G112SP1G, from LaboratorySupplies Company, Hicksville, NY was used for sonications.

Viral Growth and Purification. Virus was grown and purified essentiallyas described by Hsu et al., Virology 95, 476 (1979). Sendai(parainfluenza type I) and influenza (A/PR8/34) viruses were propagatedin the allantoic sac of 10 or 11 day old embryonated chicken eggs. Eggswere inoculated with 1-100 egg infectious doses (10³ to 10⁵ viralparticles as determined by HA titer) in 0.1 ml of phosphate bufferedsaline (0.2 gm/L KCl, 0.2 gm/L KH₂ PO₄, 8.0 gm/L NaCL, 1.14 gm/L Na₂HPO₄, 0.1 gm/L CaCl₂, 0.1 gm/L MgCl₂ 6H₂ O (pH 7.2)). Eggs wereincubated at 37° C. for 48 to 72 hours, followed by incubation at 4° C.for 24 to 48 hours. Allantoic fluid was collected and clarified at 2,000rpm for 20 min at 5° C. in a Damon IEC/PR-J centrifuge. The supernatantwas then centrifuged at 13,000 rpm for 60 min. This and all subsequentcentrifugations were performed in a Sorvall RC2-B centrifuge at 5° C.using a GG rotor. The pellets were resuspended in phosphate bufferedsaline (pH 7.2) by vortexing and sonicating, followed by centrifugationat 5,000 rpm for 20 min. The pellet was resuspended by vortexing andsonicating, diluted, and centrifuged again at 5,000 rpm for 20 min. Thetwo 5,000 rpm supernatants were combined and centrifuged at 13,000 rpmfor 60 min. The resulting pellets were resuspended in phosphate-bufferedsaline by vortexing and sonicating, aliquoted, and stored at -70° C.Sterile technique and materials were used throughout viral inoculation,isolation, and purification.

Extraction of Viral Glycoproteins and Lipids. Virus stored at -70° C.was thawed, transferred to sterile thick-walled polycarbonate tubes, anddiluted with buffer A (2 mM TES, 2 mM L-histidine, 100 mM NaCl (pH7.4)). It was pelleted at 30,000 rpm for 1 h at 5° C. in a Beckman TY65rotor. The supernatant was removed and the pellet resuspended to aconcentration of 2 mg viral protein per ml of extraction buffer (2MNaCl, 0.02M sodium phosphate buffer (pH 7.4)) by vortexing andsonicating. The nonionic detergent β-D-octylglucopyranoside was thenadded to a concentration of 2% (w/v). The suspension was mixed,sonicated for 5 sec, and placed in a 37° C. water bath for 45 min. At15, 30, and 45 min incubation times, the suspension was removed brieflyfor mixing and sonication. Nucleocapsids were pelleted by centrifugationat 30,000 rpm for 45 min in a TY65 rotor. The resulting clearsupernatant was removed and used in the formation of large, viralglycoprotein-containing vesicles. Some modification of the aboveprocedure may have to be employed with other membrane proteins. Suchmodifications are well known to those skilled in the art.

Formation of Cochleate Intermediates

A. Standard Cochleates.

Large, unilamellar, non-protein-containing, phospholipid vesicles weremade by a modification of the calcium-EDTA-chelation technique describedby Papahadjopoulos et al Biochim. Biophys. Acta 394, 483 (1975).Phosphatidylserine and cholesterol (9:1 wt ratio) were dried down in aclean glass tube under a stream of nitrogen. The lipid was resuspendedin buffer A (pH 7.4) to a concentration of 6 μMol/ml by vortexing for 7min. The resulting suspension of multilamellar vesicles was converted tosmall unilamellar vesicles by sonication under nitrogen at 5°-10° C. forapproximately 20 min in a bath-type sonicator. (Model G1125P16,Laboratory Supplies Co., Hicksville, NY). These vesicles were dialyzedat room temperature against two changes of 250 ml of buffer A (pH 7.4)with 3 mM CaCl₂. This results in the formation of an insolubleprecipitate referred to as cochleate cylinders.

B. DC Cochleates.

The envelope glycoproteins of Sendai virus account for about 33% of thetotal viral protein and are present in approximately equal weight to theviral lipid. An amount of phosphatidylserine and cholesterol (9:1 wtratio) equal to 4 times the weight of the viral lipid was dried downunder nitrogen in a clean glass tube. The amount of lipid added to theinfluenza virus extract was also equal to four times of the total viralprotein. Supernatant from octylglucoside-extracted virus (see Extractionof Viral Lipids and Glycoproteins) was added, and the solution wasvortexed for 5 min. This clear, colorless solution was dialyzedovernight at room temperature against two changes of 250 ml buffer Awith 3 mM CaCl₂. The resultant precipitate has been called DC (fordirect calcium dialysis) cochleate.

Formation of Vesicles from Cochleate Intermediates

Cochleates (DC, LC or Standard) were pelleted at 60,000×g at 5° C. for45 min. If vesicles were to be formed by the direct addition of EDTA,the supernatants were removed and replaced with a small quantity ofbuffer A containing material to be encapsulated (e.g., 50 μl buffer for10 mg of phosphatidylserine. The pellets were resuspended by vortexing.Five μl aliquots of 150 mM EDTA (pH 9.5) were added with gentle mixingand frequent monitoring of pH. The use of pH 9.5 EDTA was necessary tomaintain near neutral pH during vesicle formation. When the pH becameslightly basic, 150 mM EDTA (pH 7.4) was added until the cochleates weredissolved and an opalescent suspension of vesicles was obtained.

In the rotary dialysis method, supernatants were removed, and using asterile tipped pipetman set at 10 μl, cochleates were transferred to asmall segment of dialysis tubing (Spectrapor 4, 6 mm dry diameter). Thedialysis tubing had been boiled 2X with a solution of Na₂ CO₃, then 2Xwith distilled H₂ O, cut to the appropriate length, tied at one end, andautoclaved in distilled H₂ O just prior to use. Small aliquots of buffer(5 to 10 μl) containing material to be encapsulated were used to rinseout the tube and quantitatively transfer the cochleates to the dialysisbag. The bag was pushed firmly onto the end of a tapered glass rod whichhad been dipped in 90% alcohol and flamed. Waterproof tape and a Tygontubing "O" ring were used to further secure the bag to the glass rod.The other end of the rod was inserted in the rotary dialysis apparatus.The samples were dialyzed while rotating overnight at room temperatureagainst buffer A with 10 mM EDTA (final pH 7.4).

Biochemical Characterization of Vesicles

Proteins were determined by the modified Lowry method of Peterson,Analyt. Biochem. 83, 346 (1977). Phospholipid content was calculatedfrom inorganic phosphorous as measured by Bartlett, J. Biol. Chem. 234,466 (1959). Polyacrylamide gels were run and stained with Coomassiebrilliant blue as described by Laemmli, Nature 227, 680 (1970).Alternatively, proteins were visualized using the BioRad silver stainkit. Gels were dried under vacuum between two layers of DuPont film 215P D cellophane. A Beckman Model R-112 integrating densitometer was usedto determine the relative quantities of individual protein bands onpolyacrylamide gels.

Electron Microscopic Observations

Samples were negative-stained with 2.5% potassium phosphotungstate (pH6.7) on carbon supported formvar-coated copper grids, and then examinedon a JEOL 100Cx electron microscope.

For freeze fracture, membrane samples were fixed in 2.5% glutaraldehydein 100 mM cacodylate buffer, pH 7.2, for 2 h at 4° C. and equilibratedwith 30% glycerol in cacodylate buffer for 2 h. Samples were quenchfrozen in Freon 22 cooled with liquid nitrogen and fractured in aBalyers 360M freeze fracture apparatus. Platinum-coated,carbon-supported replicas were examined on a Hitachi HU- 11E electronmicroscope.

Light Microscopic Observation

Samples were mounted under a glass coverslip, sealed with nail polish,and examined with a Zeiss platoscope III equipped with phase contrastand epifluorescence optics. Photographs were recorded on Kodak Tri-Xfilm that was developed in Diafine.

Results Structure of the Calcium-Phospholipid Precipitate

In the calcium-EDTA chelation technique of Papahadjopoulos et al.supra., multilamellar vesicles composed of negatively-chargedphospholipids are converted into small, unilamellar vesicles bysonication. The addition of calcium to these vesicles causes theformation of a precipitate which, when analyzed by freeze fracture, isshown to consist of jellyroll-like structures called cochleatecylinders. The preferred method of the invention differs in that it usesa high salt buffer, containing detergent-solubilized phospholipid andglycoproteins which is dialyzed against calcium. In the lightmicroscope, the precipitate which forms is seen as numerous spheresapproximately one to ten microns in diameter with bumps or spikes ontheir surfaces, and free phosphatidylserine needles and cholesterolsheets. This is in marked contrast to the fine granular precipitateformed by the calcium-EDTA chelation technique. However, when theprecipitate produced in the present method is analyzed by freezefracture, it is shown to consist of cochleate cylinders.

Qualitative Protein Content of Vesicles

The protein content of vesicles was assessed qualitatively bySDS-polyacrylamide gel electrophoresis. In the case of both Sendai andinfluenza viruses, the octylglucoside extracted supernatant mainlycontains the viral envelope glycoproteins. The influenza virus extractalso contains some nucleocapsid protein which co-migrates with theneuraminidase glycoprotein in the gel system. The nucleocapsid protein,however, does not pellet with the cochleate precipitate and isconsequently greatly reduced or not present in the reconstitutedvesicles.

In addition to the glycoproteins, the Sendai virus octylglucosideextract also contains, as a minor component, a protein which co-migrateswith protein 5 of intact virions. Protein 5 has been shown to be relatedto the nucleocapsid protein. In the above preparations this proteinstains much more intensely, relative to the glycoproteins, with silverstain than with Coomassie brilliant blue. The identification of thisprotein, and information as to why it should be differentially extractedand reconstituted, await further study.

The octylglucoside insoluble pellets contain the other viral proteins.The major components of the influenza pellet are the membrane (M) andnucleocapsid (NP) proteins. The nucleocapsid protein is the majorspecies present in the Sendai virus pellet.

Recovery of Phospholipid and Protein

The quantities of protein present in the octylglucoside extractedsupernatants have been consistent with published values for the percentby weight of the virus which is glycoprotein. Small losses of proteinand phospholipid result from formation of bubbles at the solubilizationstep and from transfer losses and dialysis bag adherence at thecochleate formation and rotary dialysis steps. Approximately 70% of thetotal glycoprotein and phospholipid originally present is associatedwith the final vesicles. The 6:1 phospholipid/protein ratio of washed,unfractionated vesicles is comparable to that of the starting materials(5:1 ratio).

Density Gradient Fractionation of Vesicles

Vesicles were separated on the basis of density by flotation ondiscontinuous metrizamide gradients. Under the conditions used,phosphatidylserine:cholesterol vesicles floated to the top fraction andnon-vesicle associated protein pelleted. Proteoliposomes weredistributed according to their relative protein and phospholipidcontents. A heterogeneous population of vesicles, with respect todensity, was formed when either rotary dialysis or direct addition ofEDTA was used at the vesicle formation stage. The presence of largenumbers of vesicles in each fraction was indicated by light scattering,and confirmed by light microscopy and protein and phospholipiddeterminations.

Because of their lower density, the top and middle fractions containonly vesicle-associated protein. The amount of protein in the bottomfraction, which is vesicle-associated, versus that which is present inprotein or protein:phospholipid aggregates cannot be directly determinedby this method. It is known that there is a significant population ofhigh density vesicles in the bottom fraction because a substantialpercentage of the encapsulated volume on the gradient (measured asfluorescence of FITC-Dextran or radioactivity of ¹⁴ C sucrose in twicewashed, pelleted vesicles) is present in the bottom fraction. Also, thephospholipid must be associated with most of the protein present inorder for the density to be high enough to keep the vesicles in thebottom fraction.

That these initial gradient positions reflect stable properties of thevesicles can be demonstrated by isolation of individual fractions (top,middle, or bottom) and rebanding of these fractions on separate,identical gradients. Rebanding of vesicles from individual fractionsresulted mainly in distribution to the original gradient position, withsome redistribution to other fractions..

The Sendai viral glycoproteins are associated with vesicles of all threegradient fractions. The ratio of F to HN varies with thephospholipid:protein ratio and the method of preparation.

Sonication and Rebanding of Vesicles

Flotation of vesicles on metrizamide gradients demonstrated that theviral glycoproteins were vesicle associated. It did not distinguishbetween protein which was lipid-bilayer integrated, and that which mighthave been encapsulated within the aqueous interior of the vesicles.Evidence that F and HN were integrated in the lipid bilayer of thevesicles was obtained by the following experiment.

Sendai glycoprotein-containing vesicles andphosphatidylserine:cholesterol vesicles were made by rotary dialysis inthe presence of FITC-Dextran. The vesicles were washed twice and thenfractionated on metrizamide density gradients. The vesicles floating tothe top fraction were isolated and divided into two equal aliquots. Onealiquot was sonicated for 5 min in a bath-type sonicator and thenrefractionated on a second gradient. The other aliquot was rebandedwithout prior sonication.

Without sonication, the green-yellow FITC-Dextran loaded vesicles mainlyredistributed to the top of the gradient. In the sonicated samples, thetop vesicle band appeared white, while the bottom of the gradient was aclear, evenly diffused green. From the distribution of fluorescence itwas obtained that sonication, which caused these large vesicles to breakopen and reform as small vesicles, resulted in an almost complete lossof encapsulated material. In contrast, the phospholipid:protein ratiosof the three fractions were largely unaffected, demonstrating that theproteins were tightly associated with the lipid bilayers of thevesicles.

Fractionation of Vesicles by Column Chromatography Using SephacrylS-1000

The approximate mean size and variation of a population of phospholipidvesicles can be determined by column chromatography using SephacrylS-1000, see Nozaki et al., Science 217, 366 (1982). This technique wasused to evaluate the relative size and polydispersity of the subjectvesicles. It was also desired to separate large vesicles from tiny onesand any nonvesicle-associated, aggregated protein which might have beenpresent.

Elution of Sendai viral glycoprotein-containing vesicles,phosphatidylserine:cholesterol vesicles (9:1 weight ratio), anddelipidated, aggregated Sendai viral glyco-proteins from SephacrylS-1000 was carried out as follows. A 200 μl sample was applied to a 25.5cm (height) by 1.1 cm (diameter) column and eluted with Buffer A.Fractions of 1.3 or 0.65 ml were collected, and absorbance at 335 nm(light scattering) or 280 nm (protein absorbance) was determined.

The elution profile of Sendai viral glycoprotein-containing vesicles andinfluenza vesicles were similar. The vesicles eluted with a fairly sharpfront at the void volume, peaking slightly behind it at 8.4 ml elutedvolume. The peak broadened with a more gently declining slope in back.The vast majority of the vesicles were eluted by 13.0 ml.

Phosphatidylserine:cholesterol vesicles (9:1 weight ratio) prepared bythe calcium-induced fusion technique of Papahadjopoulos et al., supra.,exhibited a narrower, more symmetrical profile which peaked at 10.4 mland was mostly completed at 13.0 ml.

Delipidated Sendai viral glycoprotein aggregates gave a highest point at15.0 ml. The center, at 8.2 ml, corresponded to the elution peak ofbovine serum albumin which was used to determine the included volume ofthe column. Sendai virus which is 0.1 to 0.2 microns in diameter showedan elution peak at 11.7 ml.

These elution profiles indicated that the glycoprotein-containingvesicles produced by the instant method were heterogenous in size,ranging from 0.1 to several microns in diameter, with most of thepopulation being at the higher end of this range. They also indicatedthat large, glycoprotein-containing vesicles could be separated fromnonvesicle-associated, aggregated protein by elution of Sephacryl S-1000using these conditions. The protein and phospholipid content ofindividual fractions, and the fact that the plot of the absorbance at280 nm paralleled that of light scattering (OD₃₃₅) indicated that themajority of the protein was associated with vesicles.

Morphology of the Vesicles

The morphology of the glycoprotein-containing vesicles was examined bynegative staining and electron microscopy. Influenza and Sendai viralglycoprotein-containing vesicles prepared according to the method of theinvention using direct addition of EDTA at the vesicle formation step,provided peplomers on the surface of the vesicles which weremorphologically indistinguishable from those formed by the glycoproteinsintegrated in the lipid envelopes of the native virions.

The vesicles were seen to be heterogeneous in size, ranging from 0.1 to2.0 microns in diameter. These observations correlated well with thedata obtained by elution from the Sephacryl S-1000 column.

The arrangement of glycoprotein peplomers on the vesicle surfaces variedfrom dense, regular packing to sparse, irregular distribution. Thisvariation would give rise to vesicles of different densities, consistentwith the studies employing discontinuous metrizamide gradients. Theredid not appear to be any correlation between vesicle size and proteindensity; that is, a given peplomer density could be found on vesicles ofall sizes.

Biological Activity of Reconstituted Glycoproteins

The HN glycoprotein of Sendai virus and the HA glycoprotein of influenzavirus retained their ability to recognize and bind to cellular receptorswhen reconstituted into vesicles prepared by the method of theinvention.

FITC-Dextran was encapsulated within vesicles containing Sendai viralglycoproteins, or phosphatidylserine:cholesterol vesicles. The vesicleswere washed twice by dilution and pelleting, followed by fractionationon a discontinuous metrizamide gradient. Vesicles floating to the topfraction were isolated, washed, and mixed on a glass slide with humanred blood cells. The slide was rocked for several minutes and thesamples were washed by dilution and pelleting to reduce the number ofunbound vesicles present. Phase contrast and fluorescentphotomicrographs were taken of the same fields for each sample.

Sendai or influenza glycoprotein-containing vesicles which had beenseparated from nonvesicle-associated protein by Sephacryl S-1000 elutionor flotation on metrizamide density gradients showed immediate, massiveagglutination of human or chick red blood cells. In contrast,phosphatidylserine:cholesterol vesicles prepared by the calcium-EDTAchelation technique of the art did not cause hemagglutination.FITC-Dextran had been encapsulated within the interior of the vesicles.The same fields, when viewed using fluorescence microscopy, demonstratedthat Sendai glycoprotein-containing vesicles had adhered to the surfaceof the red blood cells, while plain phospholipid vesicles had not.Sendai viral glycoprotein-containing vesicles had also shownhemagglutination titers with human red blood cells which were similar tovirus, when compared on a number of particles per milligram of proteinbasis. They also agglutinated fibroblasts in suspension (mouse L929 andhuman HeLa) and efficiently associated with fibroblasts in monolayerculture. Similarly prepared phospholipid vesicles exhibited none ofthese properties.

When red blood cells which had been agglutinated at 4° C. with Sendaiviral glycoprotein-containing vesicles were incubated at 37° C., thenumber and size of the red cell aggregates decreased. This indicatedthat the neuraminidase activity of the HN glycoprotein (which wouldcleave the terminal sialic acid from the receptors, liberating thevesicle) was retained on reconstitution.

Hemolysis has been used extensively to demonstrate and investigate thefusogenic activities of viral envelope glycoproteins, see for exampleLenard and Miller, Virology 110, 479 (1981). Sendai or influenza viralglycoprotein-containing vesicles prepared by the method of the inventionexhibited hemolytic activity. The hemolyzing ability of Sendai viruswith that of reconstituted vesicles was compared.

Serial dilutions of virus or vesicles at pH 7.4 were mixed with 0.2 mlof 10% human red blood cells and incubated for 2 h on ice. The sampleswere transferred to 37° C. Ten hours later the samples were centrifugedat 1000 rpm for 5 min. The supernatants were removed, cleared ofvesicles by addition of 100 μl of 10% SDS, and the optical density at590 nm was determined.

The Sephacryl S-1000 peak was about one-tenth as active as the virus ona per weight basis. However, relatively small quantities were still ableto cause significant hemolysis. The unfractionated vesicles exhibitedlittle activity. This was probably due to the presence of aggregatedprotein which possessed binding but not significant hemolyzingcapability. The retention of the fusogenic activity of the Fglycoprotein in these vesicles was also indicated by membrane blebbingand heterokaryon formation by red blood cells and fibroblasts uponincubation with these vesicles at 37μ.

Discussion

A method for the reconstitution of membrane proteins into liposomes hasbeen disclosed. The method has been applied to the reconstitution of theenvelope glycoproteins of Sendai and influenza viruses but may also beused with other viruses as well as with membrane proteins from animal,plant, bacterial or parasitic organisms. The tight association with thelipid bilayer, the morphological appearance of the peplomers, and theretention of biological activity, all indicate that the glycoproteinsare reconstituted in a manner analoguous to that of the native virion.

The vesicle population which is formed when these viral glycoproteinsare reconstituted is heterogeneous with respect to density and size. Thevesicles can be fractionated into different density classes by flotationon discontinuous metrizamide gradients.

The Sendai viral glycoproteins are associated with vesicles of all threegradient fractions. The ratio of F to HN varies with thephospholipid:protein ratio and the method of preparation. Rotarydialysis at the vesicle formation step favors a higher proportion of Fincorporated into vesicles when compared to vesicle formation by directaddition of EDTA. The ratios of F:HN obtained with rotary dialysis morenearly reflect those of the intact and extracted viral envelope. Lowerdensity vesicles have a higher ratio of F:HN than higher densityvesicles when either method of EDTA introduction is employed.

A partial separation according to size can affected by columnchromatography using Sephacryl S-1000. Large vesicles which elute at orslightly behind the void volume can be separated fromnonvesicle-associated protein which is included. Vesicles eluting towardthe front of the peak tend to be larger than those eluting later. Thetail of the peak and following fractions contain mostly membranefragments and aggregates. The hemolytic activity of the vesicles is muchmore readily demonstrated when the vesicles eluting in the peak areseparated from the "interfering" aggregates.

An alternative, related method for reconstituting membrane proteins intoliposomes can also be employed. The first step, extraction of the viralenvelope and addition to dried down phosphatidylserine and cholesterol,is described in the Materials and Methods section. However, the solutionis next dialyzed against Buffer A (with no calcium) to form smallliposomes containing the glycoproteins. Calcium is then added eitherdirectly or by dialysis to form a precipitate. The precipitate ispelleted and treated as described to form vesicles. These liposomes havebiological activity and physical properties similar to the vesiclesdescribed above, except that they are somewhat small and have lowerencapsulation efficiencies.

The methods of the invention and the resulting protein-lipid vesicleshave a number of properties which represent a significant advance inprotein reconstitution and liposome production technology. (i) Thepreferred detergent used is β-D-octylglucoside. Due to its nonioniccharacter, functional integrity of the proteins tends to be maintained.Because of its high critical micelle concentration, it can be swiftlyand completely removed by dialysis. (ii) The vesicles are large and canefficiently encapsulate large molecules. If smaller vesicles aredesired, these can be obtained by extrusion through filters of definedpore size. (iii) A preferred embodiment of the method introduces thetechnique of rotary dialysis. This facilitates the reconstitution ofproteins which have been refractory to other methods. It also increasesthe efficiency of protein reconstitution and of encapsulation. (iv) Themethod includes the option of direct addition of EDTA. This allows forthe efficient encapsulation of small molecular weight substances whichwould be lost or diluted during a dialysis step. (v) The method isapplicable to the reconstitution of a wide variety of membrane proteins,as well as efficient encapsulation of a large number of substances. Avariety of substances, ranging in molecular mass from 342([G-¹⁴ C]sucrose) to 4.5×10⁶ daltons (nick-translated E. coli plasmid pBR322containing an insert), have been encapsulated at high efficiencieswithin these vesicles. The presence of a soluble protein at one mg perml does not interfere with the formation of these vesicles. (vi) Thesemethods do not use organic solvents, sonication, or extremes oftemperature, pressure, or pH at points where they might adversely affectthe biological activity of components to be encapsulated orreconstituted. (vii) This specific system, using biologically active,reconstituted Sendai or influenza virus glycoproteins can constitute animproved method for delivering molecules, such as nucleic acids,proteins, pharmacologically active agents in a biologically active formto the cytoplasm and the nucleus of animal cells.

We claim:
 1. A method for the efficient reconstitution of membraneproteins into large phospholipid vesicles with large internal aqueousspaces, which method comprises in combination:(A) extracting out thedesired membrane protein from a source biological material with anextraction buffer comprising a detergent; (B) mixing the extract with anegatively charged phospholipid, removing detergent and contacting witha calcium solution to form a cochleate intermediate; and (C) forminglarge phospholipid vesicles with integrated membrane protein in abiologically active state by contacting a solution of the cochleateintermediate of (B) with a calcium chelating agent, said largephospholipid vesicles being produced by a rotary dialysis procedureagainst a buffered calcium chelating agent solution as said calciumchelating agent.
 2. A method for the efficient reconstitution of a viralglycoprotein into large phospholipid vesicles with large internalaqueous spaces, which method comprises in combination:(A) extracting outthe desired viral glycoprotein from viral particles with an extractionbuffer comprising an alkyl glucoside; (B) mixing the extract with aphospholipid solution and deriving a cochleate intermediate in the formof a precipitate utilizing a direct calcium dialysis procedure; and (C)forming large phospholipid vesicles with integrated viral glycoproteinin a biologically active state by contacting the cochleate intermediateof (B) with an EDTA solution utilizing a rotary dialysis procedureagainst a buffered EDTA solution.
 3. The method of claim 2 wherein saidalkyl glucoside is β-D-octylglucopyranoside.
 4. The method of claim 2wherein said buffered EDTA solution comprises 2 mM TES, 2 mML-histidine, 100 mM NaCl and 10 mM EDTA (pH 7.4).
 5. The method of claim2 wherein said viral glycoprotein containing phospholipid vesicles areseparated from nonvesicle associated protein by a method selected fromthe group consisting of Sephacryl S-1000 column chromatography andflotation on metrizamide density gradients.
 6. A method for theefficient reconstitution of membrane proteins into large phospholipidvesicles with large internal aqueous spaces, which method comprises incombination:(A) extracting out the desired membrane protein from asource biological material with an extraction buffer comprising adetergent; (B) mixing the extract with a negatively chargedphospholipid, removing detergent and contacting with a calcium solutionto form a cochleate intermediate, said cochleate intermediate beingderived in the form of a precipitate utilizing a direct calcium dialysisprocedure involving removing detergent simultaneously with saidcontacting with said calcium solution; and (C) forming largephospholipid vesicles with integrated membrane protein in a biologicallyactive state by contacting a solution of the cochleate intermediate of(B) with a calcium chelating solution.